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Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity

Abstract

The initial stage of CRISPR–Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.

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Figure 1: Cas1 and Cas2 associate to form a complex.
Figure 2: Crystal structure of the Cas1–Cas2 complex.
Figure 3: Disruption of complex formation affects spacer acquisition in vivo.
Figure 4: Complex formation is required for CRISPR DNA recognition.

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Acknowledgements

We are grateful for the input on this work provided by members of the Doudna laboratory. We thank S. Floor, A.S. Lee, H.Y. Lee, R. Wilson, R. Wu and K. Zhou for technical assistance, the 8.3.1 beamline staff at the Advanced Light Source and A. Iavarone (University of California, Berkeley) for MS. We thank D. King (Howard Hughes Medical Institute, University of California, Berkeley) for Flag and HA peptides. This project was funded by a US National Science Foundation grant to J.A.D. (no. 1244557). J.K.N. and A.V.W. are supported by US National Science Foundation Graduate Research Fellowships and J.K.N. by a University of California, Berkeley Chancellor's Fellowship. P.J.K. is supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. J.N. is supported by a Long-Term Postdoctoral Fellowship from the Human Frontier Science Program Organization. J.A.D. is supported as an Investigator of the Howard Hughes Medical Institute.

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Authors

Contributions

J.K.N. performed the protein purification, biochemical and crystallography experiments. X-ray diffraction data were collected by J.K.N., P.J.K. and J.N., and structure determination was performed by J.K.N. and P.J.K. A.V.W. assisted J.K.N. with in vivo acquisition and immunoprecipitation assays. C.W.D. performed and analyzed analytical ultracentrifugation experiments. J.K.N. and J.A.D. designed the study, analyzed all data and wrote the manuscript.

Corresponding author

Correspondence to Jennifer A Doudna.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vivo acquisition with epitope-tagged Cas1 and Cas2 and in vitro reconstitution of the complex.

(a) Agarose gel of acquisition assays in BL21-AI cells overexpressing Cas1 and Cas2 with or without epitope tags. Lanes labeled ‘starter’ indicate the starter culture before inoculation into cultures in inducing (+) or non-inducing (-) conditions. The Cas1-FLAG and Cas2-HA constructs were used for the immunoprecipitation and DNA affinity precipitation experiments in this study. (b) An overlay of the c(s) distributions of Cas1 only (solid black), Cas2 only (dotted) and Cas1–Cas2 complex (solid blue). (c) AUC data table highlighting the s-values and the calculated apparent molecular weights. (d) Gel filtration chromatogram of pre-incubated, separately purified Cas1 and Cas2, as described in the Methods section. The arrow points to the expected elution peak of Cas1 dimer, based on our protein purification. (e) Coomassie-stained SDS-PAGE of fractions corresponding to the two peaks in (b). The green arrow points to Cas1 (33.7 kDa) and the orange arrow points to Cas2 (11.6 kDa).

Supplementary Figure 2 The two Cas1 dimers are structurally similar.

(a,b) Two views of the superposition of the Cas1a-b dimer (teal and blue) with the Cas1c-d dimer (gray). The root-mean-square deviation for the C-alpha backbone is 0.394. (c) The Cas1c–Cas2 interface is similar to the Cas1a–Cas2 interface, shown in Figure 3b. (d) Gel filtration chromatogram of purified Cas1 R252E using a Superdex 75 (16/60) size exclusion column.

Supplementary Figure 3 Cas1–Cas2 complex formation is required for CRISPR DNA binding.

(a) A schematic of the biotinylated DNA affinity precipitation experiments conducted in this study, as further described in the Methods section. (b) Western blot of the fractions throughout the experiment using magnetic Streptavidin beads, as opposed to streptavidin-agarose resin shown in Fig. 4e. (c) A schematic of the six 125-bp, biotinylated DNA substrates with scrambled regions within the leader sequence, shown in gray. Substrate 6 has a random DNA sequence upstream of the repeat with no similarity to the leader sequence (d) Western blot to detect Cas1 levels in the elution fractions of DNA affinity precipitations using the six biotinylated DNA substrates in BL21-AI cells overexpressiong Cas1 and Cas2. (e) Elution samples of DNA affinity precipitations in lysates from BL21-AI cells overexpressing the indicated Cas1 and Cas2 mutants. The +/- annotations are results from Fig. 3.

Supplementary Figure 4 Structure-based Cas1 and Cas2 alignments.

The E. coli Cas1 (a) or Cas2 (b) protein was aligned to its respective homologs for which crystal structures are available, as described in the Methods section. The number annotations at the C-termini refer to the last amino acid residue resolved in the structure, followed by the last residue of the full-length protein. The secondary structure cartoon is for the E. coli protein and the annotated residues (red) refer to the ones that were mutated in this study. The BLOSUM62 score conservation threshold is set to 50%, as reflected by the blue colors of the alignment. (c) Two views of a structure alignment of the Cas2 in the Cas1–Cas2 complex with all the available crystal structures of Cas2 homologs (all shown in monomers).

Supplementary Figure 5 Uncropped western blots of immunoprecipitation and DNA affinity precipitation assays.

(a) Related to Fig. 1c. (b) Related to Fig. 3f. (c) Related to Fig. 4e. (d) Related to Fig. 4f. (e) Related to Fig. 4g. (f) Related to Fig. 4h. (g) Related to Fig. 4h. (h-j) Related to Supplementary Fig. 3.

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 14926 kb)

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Nuñez, J., Kranzusch, P., Noeske, J. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat Struct Mol Biol 21, 528–534 (2014). https://doi.org/10.1038/nsmb.2820

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